The Mechanisms of cross-presentation of a long-lived viral protein

几种病毒衍生的蛋白质在感染细胞中是稳定和长寿命的，并且仍然产生细胞毒性T淋巴细胞（CTL）的免疫显性T细胞表位。最近，我们已经解决了这个谜的核蛋白（NP）的淋巴细胞性脉络丛脑膜炎病毒（LCMV），并证明，NP 118表位只能提出H-2Ld I类分子，只要NP从头合成，但不能持续从降解的预先存在的和长寿命的，成熟的NP。在这个项目中，我们想研究一个非常长寿的蛋白质，如LCMV-NP如何可以通过巨噬细胞和树突状细胞交叉呈递。我们最近表明，对于交叉呈递，成熟NP蛋白以及HSP 90家族分子伴侣的活性是LCMV-NP交叉呈递所需的。我们将调查，在什么样的步骤，在交叉介绍热休克蛋白90的活动是必需的，是否热休克蛋白90和NP相互作用，以及其他分子伴侣和蛋白酶参与LCMV-NP的交叉介绍。有趣的是，坏死抗原供体细胞的上清液含有NP交叉呈递所需的热不稳定因子。该因子将通过质谱法纯化、表征和鉴定。通过LCMV-NP包被微粒的基于UV的交联，我们将尝试鉴定在巨噬细胞和树突状细胞内吞后参与LCMV-NP加工的新蛋白。

Several virus-derived proteins are stable and long-lived in infected cells and nevertheless give rise to immunodominant T cell epitopes of cytotoxic T lymphocytes (CTL). Recently we have addressed this enigma for the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV) and demonstrated that the NP118 epitope can only be presented on H-2Ld class I molecules as long as the NP is synthesized de novo but can not be sustained from degradation of pre-existing and long-lived, mature NP. In this project we would like to investigate how a very long-lived protein like LCMV-NP can be cross-presented by macrophages and dendritic cells. We have recently shown that for cross-presentation the mature NP protein as well as the activity of HSP90 family chaperones are required for LCMV-NP cross-presentation. We will investigate, at what step in cross-presentation HSP90 activity is required, whether HSP90 and NP interact, and what other chaperones and proteases are involved in the cross-presentation of LCMV-NP. Interestingly, the supernatant of necrotic antigen donor cells contains a heat labile factor that is required for NP cross-presentation. This factor will be purified, characterized, and identified by mass-spectrometry. By UV-based cross-linking of LCMV-NP coated microparticles, we will try to identify new proteins that are involved in LCMV-NP processing after endocytosis by macrophages and dendritic cells.